The present invention relates generally to flow cytometers. More particularly, the present invention relates to optical detection systems for flow cytometer systems.
Flow cytometry is a technique that is used to determine certain physical and chemical properties of microscopic biological particles by sensing certain optical properties of the particles. Flow cytometry is currently used in a wide variety of applications including hematology, immunology, genetics, food science, pharmacology, microbiology, parasitology and oncology.
In flow cytometry, the microscopic biological particles of a sample fluid are arranged in single file in a core stream, typically using hydrodynamic focusing. The particles are then individually interrogated by an optical detection system. The optical detection system provides a light beam, which is scattered by each particle to produce a scatter profile. The scatter profile is analyzed by measuring the light intensity at both small and larger scatter angles. Certain physical and/or chemical properties of each particle can then be determined from the scatter profile.
Conventional cytometer systems use a single light source such as a laser to interrogate each particle. The light beam is often focused to an elongated shape that covers the uncertainty in particle position due to misalignment and variations in the width of the core stream. A limitation of using a single light source is that the particle position and variations in the width of the core stream cannot be directly detected. Misalignments in particle position and variations in the width of the core stream can be indicators of improper core formation. Because there may be no direct way of monitoring the characteristics of the core stream, improper core formation may go undetected.
This limitation may be further compounded because the single laser source configuration often does not provide a constant illumination intensity across the flow channel. As such, particles that pass more toward the edge of the core stream may not be as illuminated as particles that pass near the center. As a result, the sensitivity and accuracy of the system may vary depending on the lateral position of the particle through the focused elongated shape beam. Since there may be no easy way of detecting the lateral position of each particle, the variations in sensitivity and accuracy may go undetected.
Another limitation of using a single light source is that the velocity of each particle cannot be directly determined. Particle velocity is often an important parameter in estimating the particle size from light scatter signals. In conventional flow cytometry systems, the velocity of each particle is extrapolated from the pump flow rates. Accordingly, to accurately gauge the velocity of each particle, the pumps must be very precise, the tolerance of the cytometer flow chambers must be tightly controlled, no fluid failures such as leaks can occur, and no obstructions such as microbubbles can be introduced to disturb the flow or core formation. Satisfying these constraints can add significant complexity and cost to the flow cytometer system.
The present invention overcomes many of the disadvantages of the prior art by providing an optical detection system that uses two or more light sources positioned laterally at different distances from a central axis of a flow stream for providing light through different parts of the flow stream. One or more lenses are used to focus the light from the two or more light sources through the flow stream and onto a common focal point or region on the opposite side of the flow stream. One or more light detectors are then placed at, near or around the common focal point or region. A processor or the like may then receive at least one output signal from the one or more light detectors to analyze and determine selected characteristics of the flow stream.
In one illustrative embodiment of the present invention, an array of light sources and an array of lenses are used to illuminate a flow stream. To focus the light from each of the light sources through the flow stream to a common focal point or region on the opposite side of the flow stream, the pitch of the lens array is slightly different than the pitch of the light source array. This creates an offset between the optical axis of each lens and the corresponding light source, and this offset varies across the arrays. The various offsets are preferably set so that each lens focuses the light from the corresponding light source onto the common focal point or region on the opposite side of the flow stream. A multiple annular zoned detector is then positioned at, near or around the common focal point or region to measure the incident intensity distribution over various angular zone regions.
Blood cells or other particles present in the flow channel tend to diffract or scatter the light out of the central zone of the annular zoned detector and onto outer annular detector zones. Analysis of the signal strength produced by the various annular zones can be used to determine certain physical and/or chemical properties of each particle passing through the flow channel. Such an analysis can be used to determine, for example, if a particle is present in the flow stream, the speed and alignment of the particle within the flow stream, and in many cases, the type of particle.
In one illustrative application, the optical detection system of the present invention may be used in conjunction with a portable cytometer system for detecting, for example, neutrophils and/or lymphocytes white blood cells in a blood sample. By examining the scatter distribution of each of the particles, the portable cytometer may identify and count the neutrophils and lymphocytes in the blood sample, and provide a clear infection warning with differentiation between viral and bacterial causes. Many other applications are also contemplated.